The purpose of the experiments proposed in this application is to determine the structural characteristics and timeline of functional maturation of human olfactory cells in vitro. Mature olfactory neurons in vivo are readily identified by morphology, molecular markers, and the capability to respond to odorants; their ability to regenerate throughout the adult lifespan makes olfactory tissue an ideal system for cell culture studies. In identifying olfactory neurons in vitro, however, there appear to be dichotomous perspectives on the precise criteria of a "mature" cell: should it be based on the presence of molecular markers that signal maturation in vivo, or on the ability of cells to respond to odorant stimulation? Previous studies have primarily focused on the expression pattern of marker molecules (= "structural maturation"). This project will focus on understanding the development of functional characteristics of human olfactory neurons in vitro by determining whether the acquisition of odorant sensitivity is related to the expression of the marker molecules that signal maturation in vitro and in vivo. Cells will be grown in vitro using cell culture techniques. On each day from one to six days after a batch of cells is dissociated or "passaged" from the original stock, a subset of cells will be tested for their odorant sensitivity, with calcium imaging techniques, using a broad array of odorant mixtures. These same cells will subsequently be tested with immunocytochemical techniques to determine the pattern of expression of molecular markers found in odorant-sensitive and -insensitive cells. These experiments will quantify both the time course of functional maturation and the co-occurrence of marker molecule expression with the acquisition of odorant sensitivity. This project is important since few studies have investigated the development of neuronal function in the olfactory system, and none have correlated the presence of specific molecular markers with olfactory function. By understanding functional maturation, it will be possible to identify candidate molecules that may guide the development and maturation of olfactory neurons in vivo, identify important target molecules for pharmacological investigations of medical treatments of problems related to olfactory dysfunction and/or neurodegenerative disorders, and further the development of in vitro models for the study of human olfaction.